Identification of New hmwA Alleles from Nontypeable Haemophilus influenzae

Abstract

High-molecular-weight proteins of Haemophilus influenzae mediate attachment to epithelial cells. Previous reports describe several allelic versions of hmwA genes that have different adherence properties. Here we report three new alleles of hmwA (hmwA from strain AAr96, hmwA from strain AAr105, and hmwA from strain G822), demonstrating the high degree of DNA variation of these genes among different strains.Nontypeable Haemophilus influenzae (NTHi), a gram-negative, nonencapsulated, human-specific microorganism, commonly inhabits the upper respiratory tract and causes otitis media, conjunctivitis, sinusitis, pneumonia, and acute exacerbation of chronic bronchitis. Occasionally, NTHi causes severe invasive diseases, such as meningitis, endocarditis, and bacteremia (14).NTHi high-molecular-weight (HMW) proteins mediate bacterial attachment to epithelial cells (7,11,17,18) and have been implicated as possible virulence factors for otitis media (12,20) or chronic obstructive pulmonary disease (21). This adhesin is produced by the action of three genes (hmwA, hmwB, and hmwC) located in the hmw locus (1, 2). Many H. influenzae strains contain two distinct hmw loci, hmw-1 and hmw-2 (3, 4). The hmwA genes of these loci encode the HMWA adhesive proteins, which are 52 to 62% identical at the amino acid level (4) among several NTHi strains. hmwB genes encode outer membrane translocator proteins, which are 99% identical in NTHi strain 12. HMWB proteins are located in the outer membrane and serve to translocate HMWA across the outer membrane and prevent degradation by periplasmic proteases. hmwC genes encode cytoplasmic proteins, which are 97% identical in NTHi strain 12, and appear to stabilize HMWA (2, 17) and to influence glycosylation of HMWA1 (10). HMWA1 of strain 12 mediates binding to ␣-2,3-linked sialylated glycoproteins, and the epithelial cell receptor structure for HMWA2 of this strain is unknown (17).While initial studies of hmwA described the alleles hmwA1 and hmwA2, one in each hmw locus of strain 12 (3), Van Schilfgaarde et al. (21) described a third hmwA allele from the chronic obstructive pulmonary disease H. influenzae strain A950006, whose 4,671-bp gene encodes a predicted protein with 70% homology to HMWA1 and 68% homology to HMWA2 of strain 12. More recently, Buscher et al. (4) have identified four additional hmwA alleles with either HMWA1-or HMWA2-like binding characteristics from two NTHi strains and showed that the genes encoding these differential binding characteristics were variably located downstream of either HI01679 or HI01598 in the Rd genome.Although type b strains lack hmw loci, 55 to 80% of NTHi strains have these genes (1,8,20). Among the other encapsulated H. influenzae types, hmw loci were detected in 26% of type a, 8% of type e, and 5% of type f strains (16). In a previous study by our group using dot blot hybridization, 51% of NTHi isolates hybridized with strain 12 hmwA1-specific probes, 23% hybridized with hmwA2-specific probes, and 48% hybridized with hmwC-specific probes (8). While 18...

Conservation and Diversity of HMW1 and HMW2 Adhesin Binding Domains among Invasive Nontypeable Haemophilus influenzae Isolates

“…In comparison with strain 12 sequences, both the hmw1A and hmw2A genes from strain 72 displayed high conservation at the 5Ј end of the gene and at the 3Ј end, while the middle portion exhibited considerable genetic diversity as a result of multiple point mutations, insertions, and deletions. These data confirmed that the middle portion of the hmwA genes diverges not only between the hmw1A and hmw2A genes from strain 12 but also among each hmw1A gene and among each hmw2A gene from different isolates (9).…”

“…On the other hand, sequence analysis of hmwA alleles from different isolates revealed a high level of polymorphism in the receptor binding regions (9). In this study, a PCR approach in conjunction with DNA sequencing was used to investigate the conservation and diversity of the HMW1 and HMW2 binding domain sequences among isolates.…”

Histidine Auxotrophy in Commensal and Disease-Causing Nontypeable Haemophilus influenzae

“…Several host and epidemiological factors play a role in otitis media pathogenesis, including genetic predisposition, preceding viral respiratory infections, attendance in day care centers, lack of breastfeeding, and young age (7,12,48,55). Recent data suggest that bacterial virulence factors also play a role in otitis media (40), such as lic2B, which is involved in lipooligosaccharide biosynthesis, and the hmw genes, which encode highmolecular-weight adhesins (17,46). These two genes were shown to be more prevalent in middle ear isolates than in throat strains from healthy children, suggesting their role in otitis media virulence.…”

Antibodies Specific for the High-Molecular-Weight Adhesion Proteins of NontypeableHaemophilus influenzaeAre Opsonophagocytic for both Homologous and Heterologous Strains

“…As might have been predicted, the opsonophagocytic activity of a given immune serum was in all instances as active or more active against the respective homologous strain than were any of the other immune sera (Table 2). However, despite the known variability in the predicted amino acid sequences of the HMW1/HMW2-like proteins from different nontypeable Haemophilus influenzae strains (9,18,23), the immune sera were frequently able to mediate opsonophagocytic killing of heterologous strains. As a case in point, the predicted amino acid sequences of the HMW1/HMW2-like proteins of strains 12 and 15 demonstrated between 65 and 81% sequence identity when the four sequences were aligned and compared (18,60).…”

“…At the extreme was nontypeable Haemophilus strain 14, a strain resistant to killing by all of the immune sera that we had prepared. While strain-tostrain variation in the amino acid sequences of the HMW1/ HMW2-like proteins undoubtedly accounts for some of the differences observed (9,18,23), other explanations also deserve consideration. The bacterial surface of nontypeable Haemophilus influenzae is known to be a very dynamic structure.…”